5-ALA治疗和hTERT mRNA的反义寡核苷酸联合应用对人卵巢癌细胞的杀伤作用及其机制

doi:10.7694/jldxyxb20130608

吉林大学学报(医学版)

5-ALA治疗和hTERT mRNA的反义寡核苷酸联合应用对人卵巢癌细胞的杀伤作用及其机制

魏晓强1^,2，唐猛²，杨绍文²，张友忠¹

(1．山东大学齐鲁医院妇科，山东济南 250012;2. 青岛大学医学院第二附属医院胸外科，山东青岛 266042)

收稿日期:2013-05-08 发布日期:2013-11-28
通讯作者: 张友忠 E-mail:（Tel:0531-22166675,E-mail：manuscription@163.com)
作者简介:魏晓强（1978-），女，山东省淄博市人，主治医师，医学博士，主要从事宫颈癌的基础和临床研究。 
基金资助:
国家自然科学基金资助课题（30772327）

Killing effect of 5-ALA photodynamic therapy combined with hTERT mRNA　antisense oligodeoxynucleotides on human ovarian carcinoma cells and its mechanism


WEI Xiao-qiang^1,2,TANG Meng²,YANG Shao-wen²,ZHANG You-zhong¹

(1. Department of Gynecology,Qilu Hospital,Shandong Univeristy,Jinan 250012,China;2. Department of Thoracic Surgery,Second Affiliated Hospital,College of Medical Science,Qingdao University,Qingdao 266042,China)

Received:2013-05-08 Published:2013-11-28

摘要/Abstract

摘要：

目的：观察5-氨基乙酰丙酸（5-ALA）光动力治疗（PDT）和人端粒酶催化亚单位 (hTERT) mRNA的反义寡核苷酸（ASODN)联合应用对人卵巢癌3AO细胞的增殖抑制及促凋亡作用，探讨两种方法联合应用协同促进对人卵巢癌3AO细胞的杀伤作用机制。方法：采用MTT法筛选不同浓度5-ALA 和激光能量照射对人卵巢癌3AO细胞增殖抑制作用的最佳组合参数； RT-PCR法检测hTERT ASODN和SODN转染后hTERT mRNA表达水平的变化。将人卵巢癌3AO细胞分为空白
对照组、hTERT ASODN转染组、hTERT SODN转染组、5-ALA PDT组、hTERT ASODN转染+5-ALA PDT组和hTERT SODN转染+5-ALA PDT组，应用MTT法检测各组人卵巢癌3AO细胞增殖抑制率；应用膜联蛋白V-硫氰酸荧光素(AnnexinV-FITC)/碘化丙啶(PI)双染色结合流式细胞术检测
各组人卵巢癌3AO细胞凋亡率。结果：5-ALA PDT对人卵巢癌3AO细胞的增殖抑制作用随着光敏剂浓度的增加和激光能量的增大而增加(P<0.05)；不同浓度hTERT ASODN转染人卵巢癌3AO细胞24 h后，hTERT mRNA表达水平呈浓度依赖性下调(P<0.05)，而hTERT-SODN对人卵巢癌3AO细胞hTERT mRNA表达无显著影响(P>0.05)。hTERT ASODN转染+5-ALA PDT (5-ALA浓度为0.5 mmol•L-1，激光能量密度为2.50 J•cm-2)组人卵巢癌3AO细胞的增殖抑制率高于5-ALA PDT组和hTERT ASODN组(P<0.05)。hTERT ASODN转染+5-ALA PDT组人卵巢癌3AO细胞的凋亡率为29.28％，显著高于hTERT ASODN 转染组（12.79％）和5-ALA PDT组（21.19％)(P<0.05）。结论：hTERT ASODN转染与5-ALA PDT联合治疗可协同增强对人卵巢癌3AO细胞的增殖抑制作用，其机制可能与联合作用促进细胞凋亡有关联。

关键词: 5-氨基乙酰丙酸, 光动力治疗, 人端粒酶催化亚单位, 反义寡核苷酸, 联合治疗

Abstract:

To observe the effect of 5-ALA photodynamic therapy (5-ALA　PDT) in combination with human telomerase reverse transcriptase mRNA antisense oligodeoxynucleotides (hTERT ASODN) on proliferation inhibition and apoptosis of human ovarian carcinoma 3AO cells,and to disscuse the mechanism of the killing effect of the combination treatment on human ovarian carcinoma 3AO cells.Methods MTT assay was used to screen he best appropriate combined parameters of different concentrations of 5-ALA and laser doses on proli
feration inhibition of human ovarian cancer 3AO cells;the expression changes of hTERT mRNA were detected by RT-PCR after transfected with hTERT ASODN and sense oligodeoxynucleotides (SODN).Then human ovarian carcinoma 3AO cells were divided into blank control group,hTERT ASODN transfection group,hTERT SODN　transfection group,5-ALA PDT group,hTERT ASODN transfection combined with 5-ALA
PDT group,and hTERT SODN transfection combined with 5-ALA PDT group.The inhibitory rates of proliferation of human ovarian carcinoma 3AO cells we
re detected by MTT assay;the apoptotic rates of human ovarian carcinoma 3AO cells were conducted with Annexin-V/PI and flow cytometry.Results The inhibitory rate of proliferation of 5-ALA PDT on human ovarian carcinoma 3AO cells was dependent on the concentrations of photosensitizer and laser doses(P<0.05).The 24 h transfection in human ovarian carcinoma 3AO cells with different concentrations of hTERT ASODN diminished the abundance of hTERT mRNA in a concentration-dependent manner(P<0.05) and hTERT SODN had no effect on the expression of hTERT mRNA(P>0.05).The inhibitory rate of proliferation of cells in hTERT ASODN transfection combined with 5-ALA PDT (0.50 mmol•L-1 5-ALA,2.50 J•cm-2 laser dose of PDT) group was significantly increased compared with 5-ALA PDT group and hTERT ASODN transfection group(P<0.05).The apoptotic rate of human ovarian carcinoma 3AO cells in hTERT ASODN transfection combined with 5-ALA PDT group was 29.28%,which was significantly higher than those in hTERT ASODN transfection group(12.79%) and 5-ALA PDT group (21.19%)(P<0.05).
Conclusion The administration of 5-ALA PDT combinated with hTERT ASODN display synergistic proliferation inhibition on human ovarian carcinoma 3AO cells,and the mechanism may be related to promoting apoptosis.


Key words: 5-aminolevulinic acid, photodynamic therapy, human telomerase reverse transcriptase, combination treatment〖WT〗

中图分类号:

R737.31

魏晓强，唐猛，杨绍文，张友忠. 5-ALA治疗和hTERT mRNA的反义寡核苷酸联合应用对人卵巢癌细胞的杀伤作用及其机制[J]. 吉林大学学报(医学版), 2013, 39(6): 1121-1126.

WEI Xiao-qiang,TANG Meng,YANG Shao-wen,ZHANG You-zhong. Killing effect of 5-ALA photodynamic therapy combined with hTERT mRNA　antisense oligodeoxynucleotides on human ovarian carcinoma cells and its mechanism
[J]. Journal of Jilin University Medicine Edition, 2013, 39(6): 1121-1126.

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5-ALA治疗和hTERT mRNA的反义寡核苷酸联合应用对人卵巢癌细胞的杀伤作用及其机制

Killing effect of 5-ALA photodynamic therapy combined with hTERT mRNA　antisense oligodeoxynucleotides on human ovarian carcinoma cells and its mechanism


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5-ALA治疗和hTERT mRNA的反义寡核苷酸联合应用对人卵巢癌细胞的杀伤作用及其机制

Killing effect of 5-ALA photodynamic therapy combined with hTERT mRNA antisense oligodeoxynucleotides on human ovarian carcinoma cells and its mechanism 

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Killing effect of 5-ALA photodynamic therapy combined with hTERT mRNA　antisense oligodeoxynucleotides on human ovarian carcinoma cells and its mechanism
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